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• W1839942394 abstract "Article31 July 2012free access Source Data Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM Yi-Min Wu Yi-Min Wu Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Jen-Wei Chang Jen-Wei Chang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chun-Hsiung Wang Chun-Hsiung Wang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yen-Chen Lin Yen-Chen Lin Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Pei-lun Wu Pei-lun Wu Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shih-hsin Huang Shih-hsin Huang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chia-Chi Chang Chia-Chi Chang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Xiaopeng Hu Xiaopeng Hu School of Pharmaceutical Science, Sun Yet-Sen University, Guangzhou, China Search for more papers by this author Averell Gnatt Corresponding Author Averell Gnatt Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Wei-hau Chang Corresponding Author Wei-hau Chang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan Genomic Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yi-Min Wu Yi-Min Wu Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Jen-Wei Chang Jen-Wei Chang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chun-Hsiung Wang Chun-Hsiung Wang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yen-Chen Lin Yen-Chen Lin Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Pei-lun Wu Pei-lun Wu Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shih-hsin Huang Shih-hsin Huang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chia-Chi Chang Chia-Chi Chang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Xiaopeng Hu Xiaopeng Hu School of Pharmaceutical Science, Sun Yet-Sen University, Guangzhou, China Search for more papers by this author Averell Gnatt Corresponding Author Averell Gnatt Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Wei-hau Chang Corresponding Author Wei-hau Chang Institute of Chemistry, Academia Sinica, Taipei, Taiwan Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan Genomic Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Author Information Yi-Min Wu1, Jen-Wei Chang1, Chun-Hsiung Wang1, Yen-Chen Lin1, Pei-lun Wu1, Shih-hsin Huang1, Chia-Chi Chang1, Xiaopeng Hu2, Averell Gnatt 3 and Wei-hau Chang 1,4,5 1Institute of Chemistry, Academia Sinica, Taipei, Taiwan 2School of Pharmaceutical Science, Sun Yet-Sen University, Guangzhou, China 3Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD, USA 4Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan 5Genomic Research Center, Academia Sinica, Taipei, Taiwan *Corresponding authors: Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Tel.: +1 410 706 8239; Fax: +1 410 706 0032; E-mail: [email protected] of Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan. Tel.: +886 2 2789 8558; Fax: +886 2 2783 1237; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2012)31:3575-3587https://doi.org/10.1038/emboj.2012.205 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co-activator. Here, we employed single-particle cryo-electron microscopy (cryo-EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ∼19 Å. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf-Rpb1 jaw, supported by antibody labelling experiments. These structural features correlate with the moderate increase in the efficiency of RNA chain elongation by RNAP II(G). In addition, our updated RNAPII–TFIIF map showed that TFIIF tethers multiple regions surrounding the DNA-binding cleft, in agreement with cross-linking and biochemical mapping. Gdown1's binding sites overlap extensively with those of TFIIF, with Gdown1 sterically excluding TFIIF from RNAPII, herein demonstrated by competition assays using size exclusion chromatography. In summary, our work establishes a structural basis for Gdown1 impeding initiation at promoters, by obstruction of TFIIF, accounting for an additional dependent role of Mediator in activated transcription. Introduction RNA polymerase II (RNAPII) synthesizes all eukaryotic messenger RNA and constitutes the core of the protein encoding transcription machinery. Regulation of RNAPII transcription is crucial for cell growth and differentiation and is achieved by the concerted activities of a large number of proteins. As RNAPII is unable to recognize a promoter, promoter-specific initiation by RNAPII requires a set of conserved general transcription factors (GTFs), TFIIB, TFIID, TFIIF, TFIIE, and TFIIH to form a pre-initiation complex (PIC) at a promoter (Hahn, 2004). RNAPII was originally isolated as a multisubunit enzyme from mammalian cells (Roeder et al, 1976) but rigorous definition of its subunit composition (Rpb1–Rpb12) was facilitated by purification and characterization of its yeast counterpart (Young, 1991). The atomic coordinates of RNAPII were obtained by X-ray diffraction analysis of three-dimensional (3D) crystals grown from highly purified yeast protein (Cramer et al, 2001; Gnatt et al, 2001). The structure of RNAPII can be delineated as an assembly of distinct modules. A core module containing the active centre accounts for approximately half of the total mass of the enzyme (∼500 kDa), and three additional modules surrounding the DNA-binding cleft are mobile: the jaw-lobe (Rpb1–Rpb2), shelf (Rpb5), and clamp (Rpb1). X-ray structures of RNAPII–TFIIB (Kostrewa et al, 2009; Liu et al, 2010) along with that of the TFIIB–TBP–TATA-element ternary complex (Nikolov et al, 1995) allow for the development of a model for the PIC. As large numbers of protein contacts are involved in PIC, its formation stands as a key point of regulation (Fuda et al, 2009). Beyond PIC formation, an additional layer of regulation at the promoter requires co-activators for cell viability. Co-activator proteins convey signals from DNA-binding activators or repressors to RNAPII, allowing for up or down-regulation of gene expression. Among the co-activators, of critical importance is Mediator—an essential and conserved protein complex of ∼30 polypeptides that supports gene activation by ‘mediating’ between the activator and the PIC (Conaway et al, 2005; Kornberg, 2005; Malik and Roeder, 2005, 2010). Remarkably, an in-vitro transcription assay containing purified mammalian proteins including GTFs, general co-activator PC4, and 12-subunit RNAPII, displayed an unregulated and unfettered high degree of transcription in the presence of DNA-binding activators with or without Mediator (Hu et al, 2006). As such, it appeared that transcription factor activation of RNAPII was not dependent upon Mediator in contrast to Mediator's established role as an essential transcriptional co-activator (Belakavadi and Fondell, 2006; Casamassimi and Napoli, 2007; Cai et al, 2009). This apparent discrepancy was resolved by the disclosure of a novel RNAPII isoform, RNAPII(G), containing the RNAPII-associated polypeptide, Gdown1 of 43 kDa, which suppresses activated transcription but is relieved only in the presence of Mediator (Hu et al, 2006). Essentially, Gdown1 confers Mediator responsiveness upon RNAPII. Gdown1 is one of the many products of the GRINL1A complex transcription unit (Roginski et al, 2004) and is a novel RNAPII subunit (POLR2M) as it is resistant to dissociation from RNAPII by high salt and urea, and is found as a percent of native enzyme (Hu et al, 2006). Considering the fundamental role of transcription, and the large number of interacting transcription proteins needed for effective transcription, it is important to derive a rudimentary understanding as to how Gdown1 could crosstalk with the transcription machinery. Here, by employing cryo-electron microscopy (cryo-EM) followed by single-particle analysis, we obtained the 3D structure of RNAPII(G) in an unstained state to ∼19 Å and revealed the binding sites of Gdown1 on RNAPII. In addition, we obtained the 3D cryo-EM map of mammalian RNAPII–TFIIF and uncovered the densities of TFIIF on RNAPII and found TFIIF shared several sites with those of Gdown1. As such, the notion Gdown1 and TFIIF would exclude each other was suggested and confirmed by a gel-filtration competition assay. Our findings thus confer a steric mechanism underlying Gdown1 inhibits TFIIF function (Cheng et al, 2012; Jishage et al, 2012). Finally, the involvement of Mediator negating Gdown1 to restore transcription initiation is discussed. Results Biochemical characterization of bovine RNAPII and RNAPII(G) After receiving the native bovine RNAPII and RNAPII(G) in ammonium-sulphate precipitant, the proteins were thawed and exchanged into physiological buffer conditions. At that stage, the RNAPII and RNAPII(G) enzymes were examined for their subunit composition on a SDS–PAGE stained by Coomassie Blue. As shown in Figure 1A, the Gdown1 in the native RNAPII(G) appears to be approximately stoichiometric when compared with the two largest RNAPII subunits, with the ratio Rpb1: Rpb2: Gdown1∼0.81: 1: 0.74. Both forms of polymerase were tested for their activity in a nonspecific transcription elongation assay with tailed DNA template without the requirement of general transcription initiation factors. RNAPII and RNAPII(G) were active in generating early arrested RNA transcripts of 13–16 bases length and additional readthrough products of various lengths. Quantitation of early arrest or readthrough transcripts indicated a 1.5- to 2.5-fold increase in the amount of transcripts by RNAPII(G) compared with those of RNAPII (Figure 1B). This increase in activity of RNAPII(G) compared with RNAPII was also observed by others (Cheng et al, 2012; Jishage et al, 2012). We further analysed Gdown1's propensity as a disordered protein by rendering its sequence to folding analysis (Prilusky et al, 2005). Interestingly, the major folded region of Gdown1 appears to be in the N-terminal half, ranging from amino acid 55–113 (Figure 1C). To validate such prediction, recombinant Gdown1 proteins were subjected to limited trypsin proteolysis followed by mass spectroscopy. As anticipated, the cleavage mainly took place in the C-terminal region (Figure 1D). Figure 1.Biochemical and bioinformatics characterization of RNAPII(G). (A) Purification of native RNAPII and RNAPII(G). Both forms of calf thymus RNAPII are presented in the SDS–PAGE Coomassie stained gel, with Gdown1 and the RNAPII subunits Rpb1, Rpb2, and Rpb3 labelled. By dividing the integrated intensity over the respective molecular weight, the relative amounts of Rpb1, Rpb2, and Gdown1 in RNAPII(G) were determined to be 0.81: 1: 0.74. (B) Nonspecific transcription elongation assays. 0.4 and 0.8 μg of RNAPII (lanes 1–2) and RNAPII(G) (lanes 4–5) were used for the assays as previously described (Gnatt et al, 1997). RNA fragments from the early arrest or read-through are marked. As a control, α-amanitin, an RNAPII inhibitor, was added to RNAPII (lane 3) and RNAPII(G) (lane 6), respectively. All six lanes were from the same blot and only irrelevant lanes have been removed for the figure. Lanes 1 and 2 correspond to lanes 1 and 2 in the source gel; lane 3 corresponds to lane 4 in the source gel and lanes 4–6 correspond to lanes 6–8 in the source gel. The source data has been uploaded for full information. (C) Folding analysis of Gdown1. Program FoldIndex was used to evaluate the folding propensity of Gdown1. Two folded domains found are marked in green and unfolded region in red. (D) Limited proteolysis assay performed with trypsin. Single letter amino acid codes in the predicted folded region are denoted in green in contrast to the red colour for those in the predicted unfolded region. Cleavage sites of peptide fragments identified by mass spectrometry are labelled by blue carets while protected protease sites are denoted by black carets. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint Single-particle analysis of native bovine RNAPII and RNAPII(G) complexes in negative stain RNAPII and RNAPII(G) display different behaviour in solution. As shown in an EM image (Figure 2A), RNAPII(G) predominantly formed monomers. By contrast, RNAPII mainly formed dimers (Figure 2D). Images of RNAPII dimeric particles or RNAPII(G) monomeric particles were aligned using the SPIDER (Frank et al, 1996) and clustered with XMIPP (Sorzano et al, 2004). 7689 RNAPII(G) particle images conferred a set of class averages resembling the 2D projections of yeast RNAPII X-ray structure (Figure 2B; Supplementary Figure 1A). By the common-line method (Penczek et al, 1996), those class averages were used to generate an initial model, which was used to guide the angular reconstruction (Penczek et al, 1994) of RNAPII(G) to obtain a volume with ∼30 Å resolution (Supplementary Figure 1B). As the EM structure of RNAPII(G) was superimposed with the 12-subunit yeast RNAPII (Armache et al, 2005; PDB: 1WCM) (Figure 2C), good agreement was found, while Gdown1 density was virtually undetected. As to RNAPII dimers, alignment and clustering of selected dimeric particle images resulted in very few numbers of different classes, indicating the dimmers had preferred orientations. A class average of the dimer images (Figure 2E) carried the feature of yeast RNAPII dimers previously identified in 2D crystal (Darst et al, 1991; Asturias et al, 1998). To investigate the interface for dimerization of RNAPII, a dimer model was built from the 3D reconstruction of a negative-stained RNAPII(G) to suit the 2D dimer class average, by which the Rpb3 or Rpb4 subunit is suggested to participate in the dimer contact (Figure 2F). That Gdown1 can break the dimer formation suggests its binding sites on RNAPII include those interfacial areas. Interestingly, antibody against the Rpb3 subunit could induce a large fraction of RNAPII (∼70%) to form monomers (Supplementary Figure 1C). As suggested by our low-resolution negative-stain EM study together with the folding analysis, Gdown1 may dwell on RNAPII in an extended form, leading us to pursue a higher quality and more detailed structure by cryo-EM. Furthermore, though native RNAPII and RNAPII(G) appear relatively stoichiometric, we were unable to achieve a complete separation of native RNAPII(G) so that some amounts, though very minimal, of 12-subunit RNAPII certainly contaminate our RNAPII(G) preparation. Therefore, for the cryo-EM study, we generated RNAPII(G) particles by reconstituting RNAPII with a three- to four-fold excess of recombinant Gdown1 (Hu et al, 2006). Figure 2.Image analyses of native bovine RNAPII complexes. (A) Native bovine RNAPII(G), enriched in monomers (white circles), were preserved in negative stain (2% uranyl acetate, 50 nm bar scale). (B) Upper row: class averages of bovine RNAPII(G) particles obtained after reference-free alignment and clustering reveal renowned RNAPII features such as groove and stalk (Rpb4/7). Lower row: re-projections from the 3D reconstruction of native RNAPII(G) that best match the class averages above. (C) EM structure of the native bovine RNAPII(G) superimposed with the yeast RNAPII X-ray structure (PDB: 1WCM). (D) Native bovine RNAPII, enriched in dimers (white circles), preserved in negative stain (50 nm scale bar scale). (E) A representative class average of RNAPII dimer. (F) 3D model of the RNAPII dimer built from reconstruction RNAPII monomers. An orientation best matches the class average in (E) shows subunit Rpb3 and Rpb4 likely participate the dimer interface. Download figure Download PowerPoint Cryo-EM of bovine RNAPII elongation complex For a rigorous assessment of Gdown1 on RNAPII, it was desired to directly compare a 13-subunit RNAPII(G) with a 12-subunit RNAPII, both derived from bovine. Therefore, a cryo-EM structure of bovine RNAPII (12-subunit) reconstructed from its monomer projections was pursued. However, the dimeric form of RNAPII as opposed to the monomeric form of RNAPII(G) was not suited for a direct comparison. In an attempt to generate monomers from the bovine RNAPII dimer, the screening of salt conditions was exhausted but none of them succeeded to dissociate the 12-subunit RNAPII dimers into monomers. Interestingly, as RNAPII was supplemented with a nucleic acid scaffold (Kettenberger et al, 2004; Chen et al, 2009) (Supplementary Figure 2A), RNAPII predominantly formed monomeric particles in cryo images (Supplementary Figure 2B). The 12-subunit bovine RNAPII reconstituted with dsDNA/RNA is herein designated as the RNAPII elongation complex. We first generated a cryo-stained structure of bovine RNAPII elongation complex (Supplementary Figure 2C) by the angular reconstruction method using the negative-stained EM volume of RNAPII(G) as an initial model (Figure 2C). The cryo-stained structure of bovine RNAPII was consistent with that of the human RNAPII EM structure (EMD-1284) (Kostek et al, 2006) obtained by a similar approach (Supplementary Figure 2D). The cryo-stain reconstruction was employed to guide the angular parameters of ∼20 000 unstained cryo-EM images of bovine RNAPII elongation complex to a resolution ∼19 Å (Figure 3). As the resultant cryo-EM map of bovine RNAPII elongation complex was properly contoured according to the molecular mass of RNAPII, it gave no sporadic densities on the surface that did not belong to RNAPII but agreed nicely with that of the X-ray structure of the yeast RNAPII elongation complex filtered to the same resolution (Kettenberger et al, 2004; PDB: 1Y1W) (Figure 3). Since we did not inject any X-ray model of RNAPII for reconstructing the RNAPII cryo-EM images, the remarkable match between the cryo-EM maps of RNAPII with the X-ray structure strongly indicated that our reconstruction algorithm was reliable and could be applied to other RNAPII complexes in this study. Figure 3.Cryo-EM reconstruction of the reconstituted bovine 12-subunit RNAPII elongation complex. Three views of the cryo-EM reconstruction of bovine RNAPII elongation complex at ∼19 Å resolution (FSC 0.15). Superimposed with the EM envelop is the X-ray structure of yeast 12-subunit RNAPII (coloured ribbons) (PDB: 1Y1W). The threshold for rendering the EM reconstruction is chosen based on a molecular weight of ∼520 kDa. Download figure Download PowerPoint Cryo-EM of the 13 subunit bovine RNAPII–Gdown1 elongation complex To assure a complete stoichiometric presence of Gdown1 in the reconstituted RNAPII(G) samples, we added four-fold recombinant human Gdown1 (rGdown1) to the 12-subunit bovine RNAPII to form the RNAPII–rGdown1 complex. RNAPII–rGdown1 was previously determined to be functionally equivalent to native bovine RNAPII(G) (Hu et al, 2006). The ratio of four for reconstitution was determined by titration of rGdown1 to RNAPII followed by negative-stain EM observation to assess the minimal amount of rGdown1 required to turn the majority of RNAPII dimmers into monomeric particles (Supplementary Figure 3B). Such ratio of Gdown1 to RNAPII was found to completely inhibit promoter-specific transcription (Jishage et al, 2012). The RNAPII–rGdown1 complex is herein termed RNAPII–Gdown1. In addition, the RNAPII–Gdown1 complex was reconstituted with dsDNA/RNA to form the RNAPII–Gdown1 elongation complex, whose reconstruction would be used to compare with that of RNAPII elongation complex. The 3D cryo-EM reconstruction of RNAPII–Gdown1 (Supplementary Figure 3C) and RNAPII–Gdown1 elongation (Figure 4A) were both obtained to a resolution of 19 Å (Supplementary Figure 3E) from ∼25 000 unstained particle images respectively via the same route as for the RNAPII elongation complex. By subtracting the cryo-EM volume of RNAPII elongation complex from that of RNAPII–Gdown1 elongation complex, a positive difference map was obtained and scored based on thresholding in units of standard deviation (σ). Those densities above 5σ spread on RNAPII extensively with the majority found in the vicinity of the DNA cleft (Figure 4B); they were estimated to account for a mass of ∼40 kDa, fairly close to that of Gdown1. We divide those on the top of RNAPII into ‘a’ through ‘e’ according to where they dwell (Figure 4B). Remarkably, in region a, a bulky domain with mass of ∼6 kDa above 10σ appeared on the Rpb5 shelf and connected to the Rpb1 jaw. We named this domain Gdown1-a. To verify the localization of Gdown1 on RNAPII, antibody labelling experiments were performed. By negative-stain EM, a polyclonal antibody against Gdown1 was located near the Rpb1 jaw where the Gdown1-a is (left panel in Figure 4D). To further locate the terminal regions of Gdown1, a glutathione S-transferase (GST) was fused to the N-terminus of Gdown1 and a monoclonal antibody against GST was found also near the Rpb1 jaw (right panel in Figure 4D). Since our structural results indicated Gdown1 direct contacted the Rpb5 subunit of RNAPII, we tested if Gdown1 and Rpb5 would bind to each other with an in vitro pull-down assay. Indeed, recombinant Gdown1 and Rpb5 co-purified through two distinct affinity steps, supporting the notion that Gdown1 and Rpb5 would physically associate (Supplementary Figure 3F). Figure 4.Cryo-EM reconstruction of reconstituted bovine RNAPII–Gdown1 and antibody labelling experiments. (A) Front and top views of the cryo-EM reconstruction bovine RNAPII–Gdown1 elongation at ∼19 Å resolution (FRC 0.15) are depicted as solid deep green surface models. The threshold for rendering RNAPII–Gdown1 elongation is chosen based on a molecular weight of ∼560 kDa. (B) A positive difference map was calculated between the bovine RNAPII–Gdown1 elongation and bovine RNAPII elongation complex (grey mesh) by using volumes that were both filtered to 15 Å, and shown in yellow hue above 5 σ. The most conspicuous density above 10 σ is in the gap between the Rpb5 shelf and the Rpb1 jaw is shown in deep green. (C) A negative difference map was calculated between the bovine RNAPII–Gdown1 elongation and bovine RNAPII elongation complex and shown in pink hue. The additional densities attributed to the RNAPII elongation complex are likely to be the result of conformational changes (outer densities). (D) The right panel presents RNAPII–Gdown1 EM analysis with a polyclonal antibody against Gdown1 (gift from Dr David Price, University of Iowa, USA). From top to bottom are raw negative-stained images, images filtered to 50 Å to reveal RNAPII gross features, matching projections of RNAPII at 50 Å, and the corresponding 3D models. The left panel shows a similar analysis employing a monoclonal antibody recognizing GST, fused to the N-terminus of Gdown1. Antibody densities are encircled in the top rows, and their location denoted with arrows in the lower 3D models. Download figure Download PowerPoint Cryo-EM of bovine RNAPII–TFIIF elongation complex The structure of RNAPII–Gdown1 established herein has immediate functional implications. If common sites of association with RNAPII exist for other transcription factors, Gdown1 may poise a challenge for them to access RNAPII because Gdown1 binds to RNAPII so tightly as it does not dissociate either in high salt or in urea (Hu et al, 2006), in contrast to other transcription factors that readily dissociate from RNAPII in the presence of high salt (Cheng and Price, 2009). Based on the reasons to be described, TFIIF was thought to be susceptible. In higher eukaryotes, TFIIF is composed of a large and a small subunit, RAP74 and RAP30, and their yeast homologues are dubbed Tfg1 and Tfg2 respectively. Yeast also contains a third TFIIF subunit Tfg3 not present in other eukaryotes (Henry et al, 1994; Kimura and Ishihama, 2004). In the previous cryo-EM study using the yeast proteins (Chung et al, 2003), the Tfg2 subunit was assigned in the DNA-binding cleft, mainly inferred from the crystal structures of bacteria RNAP holoenzyme (Murakami et al, 2002; Vassylyev et al, 2002), while the Tfg1 subunit was interpreted to reside largely alongside or on the RNAPII stalk, formed by subunits Rpb4 and Rpb7. However, an alternative approach utilizing cleaving reagents mapped the residues of Tfg1 in the Tfg1/Tfg2 dimerization domain on the lobe/protrusion region of Rpb2 (Chen et al, 2007; Eichner et al, 2010). Recently, high-resolution cross-linking followed by mass spectroscopy allowed for positioning of almost all TFIIF residues on RNAPII (Chen et al, 2010), in which an extended track of Tfg1 was found on Rpb2, from the Rpb1 jaw-Rpb5 shelf to the Rpb2 lobe/protrusion. Providing that the interactions between RNAPII and TFIIF are largely conserved between yeast and mammals, RAP74—the mammalian homologue of Tfg1 would associate with mammalian RNAPII in a similar region. To resolve the controversies as to the location of TFIIF on RNAPII, we re-investigated the cryo-EM structure of RNAPII–TFIIF but in the context of mammalian proteins by reconstituting bovine RNAPII with recombinant human TFIIF. The updated RNAPII–TFIIF cryo-EM map has direct relevance to mammals and would eliminate any imprecise inference from the yeast. As the addition of recombinant TFIIF to bovine RNAPII did not turn the dimeric RNAPII into the monomeric form as effectively as Gdown1, we supplemented the RNAPII–TFIIF with nucleic acid scaffold, which is herein termed RNAPII–TFIIF elongation complex. The cryo-EM structure of the RNAPII–TFIIF elongation complex was reconstructed to a resolution of 19 Å (Supplementary Figure 4D) from ∼25 000 monomeric particle images (Supplementary Figure 4B) using the similar approach for RNAPII(G). The 3D cryo-EM envelop of RNAPII–TFIIF elongation (Figure 5A) also matches well with that of the bovine RNAPII elongation cryo-EM structure. However, by subtracting RNAPII from RNAPII–TFIIF, densities attributed to TFIIF were revealed. Those scored above 5σ mainly appeared around the DNA cleft but not in the cleft and were distributed in regions labelled ‘a’ through ‘e’, used for describing Gdown1 (Figure 5B). Regions ‘a’ to ‘c’ accord with the localization of TFIIF on RNAPII by cross-linking experiments (Chen et al, 2010); for example, ‘a’ on the Rpb1 jaw domain corresponds to the charged domain of Tfg1 (RAP74), ‘b’ around the Rpb2 lobe area encircles the dimerization domain of Tfg1–Tfg2 (RAP74–RAP30) together with the N-terminus of Tfg1 (RAP74), and ‘c’ next to the Rpb2 protrusion corresponds to the linker of Tfg2 (RAP30). To further confirm the localization of RAP74 independently by EM, the same GST strategy was employed. The GST protein fused to the N terminus of RAP74 was detected on the Rpb2 side of RNAPII, near the jaw or lobe (Figure 5D), agreeing nicely with the finding by cross-linking (Chen et al, 2010). Importantly, as soon as the positive difference ascribed to TFIIF is overlaid with that of Gdown1 (Figure 5E), it is evident that most of Gdown1's sites clash with TFIIF. The conflict occurs on the jaw/shelf (region a in Figure 5E), where Gdown1-a meets with the charged region of RAP74 (Chen et al, 2010) and also likely with part of RAP30 (Wei et al, 2001; Le et al, 2005); near the lobe/protrusion (regions b and c), where Gdown1 clashes with the N-terminus of RAP74 and the RAP74–RAP30 dimerization domain (Chen et al, 2010); and on the clamp (region e), where Gdown1 can run into RAP30, according to the X-ray study of yeast RNAPII-Tfg2 (Kornberg, 2007). Such findings strongly suggest that Gdown1 and TFIIF would mutually exclude each other from accessing RNAPII. Figure 5.Cryo-EM reconstruction of RNAPII–TFIIF. (A) Top and front views of the cryo-EM reconstruction of bovine the RNAPII–TFIIF elongation complex at a resolution of ∼19 Å (FRC 0.15). The threshold for rendering the complex is chosen based on the total mass of the complex (∼620 kDa). (B) A positive difference map in blue hue is overlaid on the RNAPII elongation complex (grey mesh). The difference was obtained by subtracting RNAPII with nucleic acids from RNAPII–TFIIF with nucleic acid, both filtered to 15 Å. (C) A negative difference map in red hue is overlaid on the RNAPII elongation complex (grey mesh). (D) RNAPII–TFIIF images with a monoclonal antibody recognizing the GST fused to the N-terminus of TFIIF. From top to the bottom are raw negative-stained images, images filtered to 50 Å to reveal RNAPII" @default.
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• W1839942394 date "2012-07-31" @default.
• W1839942394 modified "2023-10-17" @default.
• W1839942394 title "Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM" @default.
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